Chicken Glycogen Synthase Kinase 3β Suppresses Innate Immune Responses and Enhances Avian Leukosis Virus Replication in DF-1 Cells

ABSTRACT Glycogen synthase kinase 3β (GSK3β) is a widely distributed multifunctional serine/threonine kinase. In mammals, GSK3β regulates important life activities such as proinflammatory response, anti-inflammatory response, immunity, and cancer development. However, the biological functions of chicken GSK3β (chGSK3β) are still unknown. In the present study, the full-length cDNA of chGSK3β was first cloned and analyzed. Absolute quantification of chicken chGSK3β in 1-day-old specific-pathogen-free birds has shown that it is widely expressed in all tissues, with the highest level in brain and the lowest level in pancreas. Overexpression of chGSK3β in DF-1 cells significantly decreased the gene expression levels of interferon beta (IFN-β), IFN regulatory factor 7 (IRF7), Toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5), MX-1, protein kinase R (PKR), and oligoadenylate synthase-like (OASL), while promoting the replication of avian leukosis virus subgroup J (ALV-J). Conversely, levels of most of the genes detected in this study were increased when chGSK3β expression was knocked down using small interfering RNA (siRNA), which also inhibited the replication of ALV-J. These results suggest that chGSK3β plays an important role in the antiviral innate immune response in DF-1 cells, and it will be valuable to carry out further studies on the biological functions of chGSK3β. IMPORTANCE GSK3β regulates many life activities in mammals. Recent studies revealed that chGSK3β was involved in regulating antiviral innate immunity in DF-1 cells and also could positively regulate ALV-J replication. These results provide new insights into the biofunction of chGSK3β and the virus-host interactions of ALV-J. In addition, this study provides a basis for further research on the function of GSK3 in poultry.

pathways. In the insulin signaling pathway, activated Akt inhibits GSK3 kinase by phosphorylating GSK3a/b, allowing the phosphatase to act on downstream targets such as glycogen synthase, thereby converting the glycogen synthase to an activated state and promoting glycogen synthesis (3). Under nonactivated conditions for the Wnt signaling pathway, a disruption complex consisting of GSK3 coordinates the degradation of b-catenin (3). The role of GSK3 is less clear in the Notch pathway than in the Wnt pathway, and GSK3 has been shown to phosphorylate Notch intracellular domain (NICD) (5). GSK3 can decrease the level of Notch1 protein and reduce the transcriptional activity of Notch1 and Notch2 (6,7). Additionally, GSK3b-mediated phosphorylation has been reported to stabilize and activate Notch1, possibly by inhibiting the proteasomal degradation of Notch1 (8). In the Hedgehog signaling pathway, GSK3 forms a complex with another two kinases that phosphorylate Gli2/3 and cause Gli2/3 to generate a truncated form, finally translocating to the nucleus and repressing gene transcription. Moreover, in the transforming growth factor b (TGF-b) signaling pathway, GSK3 phosphorylates Smad3, resulting in degradation of the latter (9). Collectively, these signaling pathways are involved in the regulation of metabolism, apoptosis, cell cycle, cell differentiation, embryogenesis, and gene transcription (10,11), which sheds light on the important role of GSK3 in these cellular processes.
GSK3 also plays a vital role in regulating innate immunity. GSK3b has been reported to promote innate immune response by inhibiting 59-AMP-activated protein kinase (AMPK) activation (12). Some evidence suggests that the immunomodulatory role of GSK3 lies mainly in affecting type I interferon (IFN) signaling and exerting antiviral effects (13). Martin et al. reported that GSK3 negatively regulates anti-inflammatory cytokine production by macrophages through Toll-like receptor (TLR)-induced activation of the phosphoinositide 3kinase (PI3K)-Akt signaling pathway (14). Inhibition of GSK3b activity increases the nuclear binding level of cAMP response element-binding protein (CREB), thus promoting the production of interleukin 10 (IL-10) and reducing the production of IL-12 (15). In addition, GSK3 is related to the regulation of proinflammatory and anti-inflammatory responses. Activated GSK3b can promote the activation of NF-k B, which in turn leads to a proinflammatory response. Conversely, MyD88-dependent signaling pathway activation of TLR receptors can promote GSK3b inactivation to lead to an anti-inflammatory response (14,(16)(17)(18)(19)(20). Furthermore, infection with some viruses (e.g., human T-cell leukemia virus type 1 [HTLV-1]) causes increased levels of GSK3b phosphorylation and decreased levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-a), IL-8, and monocyte chemotactic protein 1 (MCP-1) (21). Interestingly, the infection of triple transgenic mice with mouse hepatitis virus (MHV) leads to significant increases in GSK3b activity and in the number of cells expressing major histocompatibility complex class II (MHC-II), CD4, and CD8, as well as in levels of proinflammatory cytokines (22), suggesting that different viruses cause different changes in GSK3b activity, but the exact detailed mechanisms still need to be investigated further. Moreover, GSK3 is also relevant to a variety of crucial life activities, such as cell cycle and differentiation, acquired immunity, and carcinogenesis (23).
In contrast to mammals, only one isoform of GSK3b is present in poultry (24). GSK3b has been extensively studied in mammals but has not yet been reported in avian species. In this study, chicken GSK3b (chGSK3b) was cloned and analyzed, and its distribution in various tissues was detected. Then, we detected the effect of chGSK3b on innate immunity and its regulation in avian leukosis virus subgroup J (ALV-J) infection. These results provide novel insights into the role of chGSK3b in regulating the innate immune mechanisms in chicken.
Tissue distribution of chGSK3b in SPF chickens. The chGSK3b transcript levels in different tissues of 1-day-old specific-pathogen-free (SPF) chickens were measured by and Anas platyrhynchos GSK3bs using DNAMAN software. Black, sequences conserved across the species; pink, protein sequence homology of $75%; light blue, protein sequence homology of $50%. Ser9 and Tyr216 are indicated by red asterisks above the sequences. (B) Prediction of protein structural domains using the SMART program. The pink bars indicate two low-complexity domains, located at amino acids 386 to 398 and amino acids 408 to 420 of chGSK3b.
GSK3b Mediates Innate Immunity and ALV Replication Microbiology Spectrum quantitative reverse transcription (qRT)-PCR with the RNA extracted from the corresponding tissues. The results showed that chGSK3b levels were relatively higher in brain, ileum, and thymus and lowest in pancreatic tissues (Fig. 3).
Expression of pCAGGS-chGSK3b-Flag in DF-1 cells. Following transfection of pCAGGS-chGSK3b-Flag into DF-1 cells, the expression of chGSK3b was detected by indirect immunofluorescence assay. The result showed that GSK3b was expressed well in DF-1 cells ( Fig. 4A and B).
chGSK3b is involved in the regulation of innate immunity. To investigate the role of chGSK3b in the chicken innate immune response, pCAGGS-chGSK3b-Flag or empty vector was transfected into DF-1 cells. The total cellular RNA was extracted from cells collected at the corresponding time and reverse transcribed for subsequent experiments (Fig. 5). Compared with the control group, the mRNA expression of IFN-b and IFN  (Continued on next page) GSK3b Mediates Innate Immunity and ALV Replication Microbiology Spectrum different degrees, and the mRNA expression of MDA5, TLR3, IRF7, and antiviral molecules (oligoadenylate synthase-like [OASL]) increased significantly in the chGSK3b-inhibited group, compared with the control group (Fig. 7). These results suggested that chGSK3b was involved and regulated chicken innate immunity. chGSK3b positively regulates the replication of ALV-J. To explore the effect of chGSK3b on virus replication of ALV-J, chGSK3b was overexpressed or inhibited in ALV-Jinfected cells to detect the change of virus replication and the expression levels of innate immune molecules. The results showed that the mRNA expression of IFN-b, IRF7, MDA5, TLR3, PKR, and MX-1 was significantly decreased in the overexpression group after viral infection, compared with the control group. The mRNA levels of IL-1b, IL-6, and IL-8 showed a trend of decreases and then increases, while the mRNA levels of TNF-a and IL-10 significantly increased after viral infection (Fig. 8). The gene expression levels of p27 and gp37 of ALV-J were significantly increased in DF-1 cells transfected with chGSK3b ( Fig. 9B and C). The expression level of p27 protein was also increased (Fig. 9D). The titer of the virus in the cell supernatant increased at each time point, as demonstrated by the 50% tissue culture infective dose (TCID 50 ) assay (Fig. 9E). These results indicated that overexpression of chGSK3b promoted the replication and proliferation of ALV-J. After inhibition of chGSK3b by siRNA, the mRNA expression of IFN-b, IRF7, MDA5, TLR3, PKR, MX-1, and OASL were significantly upregulated, compared with the control group (Fig. 10), and the gene expression, protein expression, and proliferation of the ALV-J were significantly decreased (Fig. 11), which indicated that the inhibition of chGSK3b suppressed the replication of ALV-J in DF-1 cells. All of these results demonstrated that chGSK3b had the biological function of promoting the replication of ALV-J.

DISCUSSION
GSK3b is an important multifunctional protein involved in the regulation of multiple signaling pathways, innate immunity, and inflammatory responses in mammals (3,16,25). However, the function of chGSK3b has not yet been investigated. In this study, the complete chGSK3b cDNA (GenBank accession number OP548633) was cloned for the first time; it is 1,263 bp long and encodes 421 amino acids. Results from BLAST analysis revealed that the chGSK3b gene is conserved in a variety of species. It shares    the Ser9 and Tyr216 phosphorylation sites with other species, which affects the biological function of GSK3. To investigate the tissue distribution of chGSK3b, the abundance of the chGSK3b gene in different tissues was analyzed. The chGSK3b gene expression was found to be widely distributed in different tissues. The highest expression level of chGSK3b is in brain, which is consistent with findings in mammals (26).
To further explore whether chGSK3b is involved in avian innate immunity, the chGSK3b was overexpressed in DF-1 cells. We found that the gene expression of IFNb, IRF7, TLR3, MDA5, MX-1, and PKR was significantly decreased after overexpression of chGSK3b in DF-1 cells. However, the expression of TNF-a was significantly increased, in support of the previous report that GSK3 positively regulates TNF-a production to modulate the innate immune response (27). Notably, previous reports showed that GSK3b could regulate IL-10 expression negatively (28)(29)(30), whereas chGSK3b significantly promoted IL-10 expression levels in our study, as shown in Fig. 5; this may be due to the different mechanisms of IL-10 regulation by GSK3b in different cells and species. Moreover, the results in Fig. 6 revealed that overexpression of chGSK3b could inhibit IFN-b promoter activity and reduce IFN-b expression also, which suggested that chGSK3b might be involved in IFN-associated immunity.
Many studies have reported that GSK3 is associated with regulating signaling pathways to affect viral replication. For example, the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) was shown to be dependent on GSK3-mediated phosphorylation of nucleocapsid protein (N), which is an essential process for viral replication (31). Our previous studies demonstrated that GSK3 inhibitors significantly increased the replication of ALV-J in CEF cells (32). The activation of the PI3K/Akt signaling pathway exerted an inhibitory effect on porcine epidemic diarrhea virus (PEDV) proliferation by inhibiting the activity of GSK3. It was also reported that a specific inhibitor of GSK3 could significantly increase the proliferation of PEDV but limit Tat-   (33). In addition, GSK3b affects hepatitis C virus (HCV) particle maturation and release and promotes the replication of influenza viruses (34,35). In this study, overexpression of chGSK3b gave rise to significant decreases in the gene expression levels of host antiviral molecules and increased the ALV-J replication, while the virus replication was suppressed significantly after chGSK3b inhibition by siRNA, which suggests that chGSK3b can positively regulate the ALV-J replication in DF-1 cells. It is noteworthy that ALV-J infected cells did not induce any IFN-b expression in DF-1 cells (data not shown). However, knockdown of chGSK3b resulted in a significant increase in IFN-b expression in virus-infected DF-1 cells (Fig. 10), which suggests that ALV-J-induced immunosuppression might be associated with chGSK3b. In previous reports, overexpression of GSK3b was reported to enhance the activation of IRF3 and the transcription of IFN-b by Sendai virus (SeV), and this effect is produced by GSK3b by causing oligomerization and phosphorylation of TANK-binding kinase 1 (TBK1) (36). It was also reported that GSK3b can interact directly with TBK1 (37) to promote TBK1 phosphorylation (38) and that overexpression of TBK1 in DEF cells causes activation of GSK3b (39). In this study, whether the mechanism of chGSK3b affecting ALV-J replication is also related to TBK1 needs to be clarified by further studies.
In summary, the chGSK3b gene was cloned and analyzed for the first time. We found that chGSK3b has high homology with genes from other birds and mammals and is widely expressed in different tissues. The results of biofunctional study reveal that chGSK3b is involved in regulating antiviral innate immunity in DF-1 cells and also can positively regulate ALV-J replication. In addition, the role of GSK3 in immunosuppression induced by ALV-J infection deserves further study.

MATERIALS AND METHODS
Animals, cells, and viruses. SPF chickens were purchased from Lihua Company (China). DF-1 cells were incubated at 37°C in a 5% CO 2 incubator. The ALV-J strain JS09GY3 (GenBank accession number Molecular cloning of chGSK3b. To amplify chGSK3b, primers chGSK3b F and chGSK3b R (Table 1) were designed based on the predicted chGSK3b sequence in GenBank XM_040660411.2. Total RNA was extracted from HD11 cells by the method of the FastPure cell/tissue total RNA isolation kit v2 (Vazyme, Nanjing, China) and reverse transcribed into cDNA using the HiScript III RT SuperMix for quantitative PCR (qPCR) (Vazyme), and PCR was performed to amplify the target sequence. The pCAGGS-chGSK3b-Flag vector was constructed by inserting the PCR product of chGSK3b into a pCAGGS empty vector digested by XhoI and NotI.
Biological analysis of chGSK3b. The amino acid sequence was deduced from the nucleotide sequence of the gene and analyzed by DNAMAN. The structural domain of chGSK3b was identified by the SMART program. Homology analysis of amino acid sequences was performed using MegAlign (DNAstar, USA). Phylogenetic trees of 14 different species, including birds, fish, mammals, and arthropods, based on chGSK3b were constructed using MEGA-X.
Tissue sample collection and real-time qPCR. Three 1-day-old SPF white leghorn laying hens were used for collection of tissue samples, including the skin, thymus, ileum, bursa of Fabricius, heart, rectum, spleen, muscle, glandular stomach, duodenum, cecum, cecal tonsil, lung, brain, jejunum, pancreas, liver,  GCCUACAAGGGAGCAAAUUTT 825 si-chGSK3b-825 (antisense) AAUUUGCUCCCUUGUAGGCTT and muscular stomach. All of the tissue collection procedures were reviewed and approved by the Animal Care Committee of Yangzhou University. Total RNA was extracted from these tissues and reverse transcribed into cDNA. In this experiment, absolute real-time qPCR was used to detect the expression level of GSK3b in each tissue. Three replications were performed for each group. The constructed pCAGGS-chGSK3b-Flag vector was used for 10-fold serial dilutions, and eight concentrations from 1:10 2 to 1:10 9 were used as the templates for the standard curve. The real-time qPCR was performed and the reactions were carried out using the ChamQ SYBR qPCR Master Mix (Vazyme) in an ABI 7500 system. The primers are presented in Table 1.
Western blot analysis. To verify the expression of chGSK3b-Flag in cells, DF-1 cells were transfected with pCAGGS-chGSK3b-Flag or pCAGGS empty vector using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The cells were lysed with a cell lysis buffer containing protease inhibitor cocktail (Beyotime, Shanghai, China), and the protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Vazyme). The target proteins were electrotransferred to nitrocellulose membranes (Sigma, Shanghai, China) after SDS-PAGE. The primary antibody was anti-GSK3b monoclonal antibody (MAb) (Cell Signaling Technology, Shanghai, China), and the secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, USA).
Dual luciferase reporter assay. The dual luciferase reporter assay kit (Vazyme) assisted in detecting gene regulation by measuring the fluorescence intensity of luciferin substrate in response to luciferase expression after transfection of cells with a reporter plasmid. DF-1 cells seeded in 12-well plates were grown to 90% confluence and cotransfected with pCAGGS-chGSK3b-Flag, reporter plasmids (pGL3-IFNb, pGL3-IRF7, and pGL3-NF-k B), and control plasmid (pRL-TK). The cells were lysed at the indicated time points for dual luciferase reporter assays with a dual-specific luciferase assay kit (Promega, Shanghai, China). Three replications were conducted for each sample.
Immunofluorescence staining. The cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized with 0.25% Triton X-100 for 10 min, and blocked with 2% bovine serum albumin in PBS for 30 min. Then, the cells were incubated with anti-GSK3b MAb (Cell Signaling Technology) in PBS for 45 min at room temperature. The cells were then washed in PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 40 min. The cells were visualized with a fluorescence microscope (Olympus IX65).
RNA interference assay. Three siRNAs targeting chGSK3b were synthesized by Suzhou Jima Pharmaceutical Technology. Then 150 pmol siRNAs or the negative control (siRNA for the negative control [siNC]) were transfected into DF-1 cells in 12-well plates using Lipofectamine reagents (Invitrogen, Shanghai, China). Total RNA was extracted for real-time PCR at 48 h posttransfection (hpt). The siRNA sequences are presented in Table 2.
Viral infection. After transfection with pCAGGS-chGSK3b-Flag and pCAGGS vector control or si-GSK3b and siNC for 24 h, the DF-1 cells were infected with the JS09GY3 strain at a multiplicity of infection (MOI) of 1.0. The cells were collected at different time points for real-time qPCR and Western blotting, and the culture supernatant was harvested for virus titer determination with the TCID 50 assay.
Data analysis. The data were expressed as mean 6 standard deviations (SD). The significance of the variability between the trials was analyzed using GraphPad Prism v8.0 software. Differences in data were evaluated with the Student t test.
Data availability. The full-length cDNA of chGSK3b was deposited in GenBank under accession number OP548633.1.